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  • A novel method for evaluating the biological activity of recombinant human interleukin-1 receptor antagonist (rhIL-1RA) produced in E. coli
  • Kianoush Dormiani,1,* pendar shojaei,2 sanaz sedghi esfahani,3
    1. 1. Department of Animal Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran
    2. Department of Animal Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Ira
    3. Department of Animal Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Ira


  • Introduction: Human interleukin-1 (hIL-1) is a pro-inflammatory cytokine that plays a critical role in controlling inflammatory responses. In the case of a continuous response, this process causes serious tissue damage and auto-inflammatory diseases such as rheumatoid arthritis. Human interleukin-1 receptor antagonist (hIL-1RA) is an antagonist of hIL-1 receptor that inhibits the signaling pathway of hIL-1 and subsequently reduces the severity of autoimmune disease. In this study, recombinant hIL-1RA protein was expressed into soluble form in E. coli. Accordingly, the present study was designed to set up a simple and accurate method to evaluate the biological activity of the recombinant hIL-1RA protein. For this purpose, NIH3T3 fibroblast cells were treated with hIL-1RA protein and after that, the process of inflammation was induced through treatment with Lipopolysaccharide (LPS). consequently, higher expression of hIL-1β reduced the cell viability of NIH3T3 cells. Then, the bioactivity of pre-treated recombinant hIL-1RA protein by antagonizing the IL-1 receptor was evaluated using MTS assay. Based on our findings, 1000 ng/ml of the recombinant hIL-1RA protein was successfully able to rescue the survival of the LPS-treated NIH3T3 cells by 82 %.
  • Methods: For the MTS assay test, NIH3T3 cells were counted and 1 × 102 cells/ml were seeded in 96-well plates. After 24 h and following cell adhesion, recombinant hIL-1RA and commercial hIL-1RA (Peprotech) were added separately at final concentrations of 10, 100, 500, and 1000 ng/ml. Two hours later, 100 µg/ml LPS was added to each well. After 24 h of LPS treatment, the cells were subjected to the MTS assay to determine the viability of NIH3T3 cells. 20 μl of the MTS/PMS reagent was added to each well and incubated at 37 °C for 3 h. At the end of incubation, absorbance was measured at 490 nm, and after normalization, the viability of NIH3T3 cells was calculated
  • Results: The results showed that there was no significant difference between the recombinant hIL-1RA activity and the commercial protein (Peprotech) as the positive control. Both of these proteins similarly neutralized the cytotoxic effect of hIL-1β and improved NIH3T3 cell viability from 82% (after treatment with LPS) to 96% (after adding hIL-1RA proteins). It is noteworthy that the rescue of cell viability by hIL-1RA proteins was dose-dependent.
  • Conclusion: Using this simple and reliable approach, the biological activity of recombinant hIL-1RA was estimated in comparison to a standard protein. This study indicated that however, treatment of NIH3T3 cells with LPS decreased the cell viability, the recombinant hIL-1RA protein was able to rescue, the cell survival, and thus the functionality of recombinant IL-1RA protein was confirmed by antagonizing IL-1 receptor and reduction of inhibitory effect of LPS on the viability of NIH3T3 cells.
  • Keywords: hIL-1, hIL-1RA, Lipopolysaccharide, NIH3T3 fibroblast cell, MTS assay